The overall goal of this proposal is to establish a targeted therapeutic strategy to disrupt the interaction of coagulation factor FXII (FXII) and urokinase plasminogen activator receptor (uPAR) to downregulate Akt2- mediated neutrophil activation for treatment of deep vein thrombosis (DVT). DVT is a leading cause of cardiovascular death. New anticoagulation therapies have been developed, however these therapeutic advances are all associated with increased rate of bleeding. Moreover, these current anticoagulants only inhibit coagulation end-points (e.g. thrombin and fibrin) but do not prevent upstream events such as neutrophil activation, procoagulant neutrophil extracellular trap (NET) formation, or propagation of neutrophil-platelet aggregates, all of which are persistent hallmark events in DVT. In this framework, we propose to use a unique nanomedicine-based therapeutic approach to downregulate neutrophil activation and NET formation through targeted disruption of the FXII-uPAR-pAkt2 axis. Our laboratory identified that FXII in neutrophils is critical for function. Specifically, we have shown that following neutrophil activation, autocrine FXII signals through uPAR leading to Akt2S474 phosphorylation (pAkt2) and NET formation. Inhibiting FXII signaling in neutrophils resulted in smaller venous thrombi. Based on these mechanistic findings, our central hypothesis is that targeted inhibition of the FXII-uPAR-pAkt2 axis will be therapeutically effective in treating DVT while minimizing systemic side- effects and bleeding risk. We will test this hypothesis by packaging uPAR inhibitory peptides within nanovesicles that are uniquely surface-engineered to undergo specific heteromultivalent anchorage onto neutrophil-platelet aggregates. In this application, our goals are to: 1) identify a candidate uPAR inhibitory peptide that disrupts FXII binding on the surface of neutrophils. We will determine the affinity, stoichiometry and specificity of inhibition, assess Akt2 activation and perform neutrophil and platelet function assays; 2) use heteromultivalently decorated nanovesicles loaded with the candidate peptide drug to determine their ability to site-selectively inhibit the FXII-uPAR interaction and mitigate DVT in vitro and in vivo; 3) to validate these preclinical studies, we will determine the constitutive activity of the FXII-uPAR-pAkt2 axis and the effect of its inhibition on neutrophil functions, neutrophil- platelet interactions and thrombus growth ex vivo, using blood samples from patients with newly diagnosed DVT. The end goal is to show the differential abundance of the FXII-uPAR-pAkt2 axis and downstream effectors in DVT pathology which will lay the foundation for future clinical studies to inhibit its action. Our scientific innovation is the mechanistic elucidation of the FXII-uPAR-pAkt2 signaling axis in neutrophil- mediated pathology. Our technological innovation is the development of inhibitory peptide-based targeted nanomedicine strategies to block this axis for therapeutic benefit in DVT. Since FXII is one of the few proteins that protects from thrombosis without increasing bleeding risk, the proposed studies have the potential to improve the benefit-to-risk profile of anticoagulant therapy in comparison with inhibition of the final common coagulation pathway that is characteristic of current anticoagulants. If successful, the proposed therapeutic strategy will introduce novel and safer therapies to treat thrombotic disorders, morbidities that are common among the Veteran population.